Peptide isolation

Abstract

A process for isolating selected polypeptides from biological specimens. The process employs a guanidinium salt to treat frozen biological specimens from which selected polypeptides are to be obtained. The process is useful in reducing proteolytic modification of the selected polypeptides.

I claim: 
     
       1. An active peptide fraction containing immunopentatiating thymic polypeptides, prepared from freshly frozen thymic tissue by a process comprising: (a) homogenenizing the frozen tissue in an aqueous cold solution containing an effective amount of a guanidinium salt, and   (b) isolating from the resulting homogenate the peptide material therein by an isolation method comprising ultrafiltration through ultrafilters having a molecular weight cut-off of 10,000 daltons and adsorption on and elution from a reverse phase hydrophobic resin with a miscible solvent capable of eluting and providing thereby the active peptide fraction.   
     
     
       2. A fraction according to claim 1 wherein the polypeptides contained by said fraction comprise thymosin β 4  and thymosin β 9 . 
     
     
       3. A fraction according to claim 1 wherein the thymic tissue is calf thymus or calf spleen. 
     
     
       4. A fraction according to claim 1 wherein the guanidinium salt is a guanidine hydrohalide. 
     
     
       5. A fraction according to claim 4 wherein the guanidine hydrohalide is guanidine hydrochloride. 
     
     
       6. A fraction according to claim 1 wherein said resin is a hydrocarbon of C 8  to C 18  on an inert matrix, said matrix being inert to said solvent. 
     
     
       7. A fraction according to claim 6 wherein the inert matrix is selected from the group consisting of silica gel, glass and polysaccharides. 
     
     
       8. A fraction according to claim 1 wherein said fraction is obtained from said resin by eluting said resin with a pyridine-formic acid buffer solution of about pH 4.0, said solution containing 40% n-propanol by volume. 
     
     
       9. An active peptide fraction containing immunopotentiating thymic polypeptides including thymosin β 4 , thymosin β 9  and prepared from freshly frozen thymus tissue by a process comprising: (1) homogenizing the tissue in the presence of an ice cold aqueous solution containing an effective amount of guanidinium salt,   (2) suspending the resulting homogenate in a pyridine-formic acid solution at about pH 4.0,   (3) centrifuging the resulting suspension and obtaining the resulting supernatant,   (4) ultrafiltering the resulting supernatant through ultrafilters having a molecular weight cut-off of 10,000 daltons,   (5) submitting the resulting filtrate to a reverse phase hydrophobic resin,   (6) washing said resin with pyridine-formic acid buffer solution of about pH 4.0, and   (7) collecting the material eluting from said resin upon applying the same buffer solution containing 40% by volume n-propanol, to provide thereby the active peptide fraction.   
     
     
       10. A process for isolating selected polypeptides from a freshly frozen biological specimen, the process comprising in sequence treating the specimen with an effective amount of guanidinium salt and separating the selected polypeptides from said specimen. 
     
     
       11. A process according to claim 10 wherein the specimen is animal tissue. 
     
     
       12. A process according to claim 11 wherein the animal tissue is calf thymus or calf spleen. 
     
     
       13. A process according to claim 10 wherein the guanidinium salt is guanidine hydrochloride. 
     
     
       14. A process according to claim 10 wherein the selected polypeptides are separated from the specimen by ultrafiltration and chromatographic methods. 
     
     
       15. A process according to claim 14 wherein the chromatographic methods comprise high pressure liquid chromatography and a reverse phase hydrophobic resin thereto. 
     
     
       16. A method for preventing and treating opportunistic infections in immunosuppressed subjects, which method comprises administering to the subject in immunopotentiating effective amount of an active peptide fraction of any one of claims 1-8. 
     
     
       17. An active peptide fraction containing immunopentiating thymic polypeptides, prepared from freshly frozen thymic tissue by a process comprising: (a) homogenizing the frozen tissue to provide a frozen powder and adding said powder to an aqueous cold solution containing an effective amount of a guanidinium salt, and   (b) isolating from the resulting homogenate the peptide material therein by an isolation method comprising ultrafiltration through ultrafilters having a molecular weight cut-off of 10,000 daltons and absorption on and elution from a reverse phase hydrophobic resin with a miscible solvent capable of eluting and providing thereby the active peptide fraction.   
     
     
       18. A fraction according to claim 17 wherein the polypeptides contained by said fraction comprise thymosin β 4  and thymosin β 9 . 
     
     
       19. A fraction according to claim 17 wherein the thymic tissue is calf thymus or calf spleen. 
     
     
       20. A fraction according to claim 17 wherein the guanidinium salt is a guanidine hydrohalide. 
     
     
       21. A fraction according to claim 20 wherein the guanidine hydrohalide is guanidine hydrochloride. 
     
     
       22. A fraction according to claim 17 wherein said resin is a hydrocarbon of C 8  to C 18  on an inert matrix, said matrix being inert to said solvent. 
     
     
       23. A fraction according to claim 22 wherein the inert matrix is selected from the group consisting of silica gel, glass and polysaccharides. 
     
     
       24. A fraction according to claim 17 wherein said fraction is obtained from said resin by eluting said resin with a pyridine-formic acid buffer solution of about pH 4.0, said solution containing 40% n-propanol by volume.